Process for producing erythromycin



United States Patent Ofiiice 2,808,363 Patented Oct. 1, 1957 Herman Hoeksema and Lionel E. Township, Kalamazoo County, Up ohn Company, Kalamazoo, Michigan No Drawing. Application June Serial No. 361,424

. 7 Claims. (Cl. 195-80) Rhuland, Kalamazoo Mich., assignors to The Mich., a corporation of This invention relates to the fermentation culturing of erythromycin ferment and is particularly directed to methods and compositions for obtaining improved yields of erythromycin characterized by the incorporating in the erythromycin substrate medium of material selected from the class consisting of propanal and n-propanol and the homologues; methanol, ethanol, and n-butanol.

It is an object of the invention to provide new and improved processes for producing erythromycin. It is another object of the invention to provide new and improved substrate media for culturing erythromycin ferment. It is another object of the invention to provide adjuvants which when added to erythromycin substrate media, function to increase the yield of erythromycin. Still other objects will appear as the description proceeds.

These objects are accomplished in the present invention by incorporating in the erythromycin substrate media used in culturing the erythromycin ferment a material selected from the class consisting of propanal and n-propanol and the homologues; methanol, ethanol, and nbutanol in an amount sufiicient to stimulate the production of erythromycin. Through the addition of said material to erythromycin substrate and culturing erythromycin ferment therein, a yield of erythromycin is obtained which is substantially greater than the yield obtainable in the absence of said material.

In carrying out the process of the invention an erythromycin ferment, such as Streptomyces erythreus NRRL 2338, is cultured in an erythromycin substrate medium according to the invention using procedures and apparatus well understood in the art. See U. S. Patent 2,653,899 and Antibiotics and Chemotherapy 2: 281- 283 (June 1952). The fermentation is carried out under aerobic conditions and advantageously by the submerged culture process. Initially the substrate medium is adjusted to between pH 6 and pH 7.5, advantageously to about 6.5. In the fermentation the pH ordinarily increases up to about pH 9. Suitably the temperature may range between about 25 degrees centigrade and about 37 degrees centigrade, advantageously between 26 and 32 degrees centigrade. In these and other respwts the procedure outlined in the above U. S. Patent 2,653,899 can be advantageously followed.

The basic erythomycin substrate is carbohydrate. Preferred sources of carbohydrate are starch and dextrose (glucose). Other sources which can be included, or substituted for starch and dextrose, are sucrose, dextrin, molasses, brown sugar, maltose, and the like. The erythromycin substrate media advantageously contain in addition, nitrogen sources, preferably such organic nitrogen sources as corn steep, soy bean meal or flour, distillers solubles, casein, amino acid mixtures, peptones (both meat and soy), brewers yeast, peanut granules, peanut meal, and the like. Inorganic nitrogen sources such as nitrate salts or ammonium salts can also be employed. The substrate media also can contain nutrient inorganic salts which furnish sodium, potassium, calcium, phosphate, chloride, sulfate, and like ions. Es-

sential trace elements such as cobalt, magnesium, iron, and manganese can be added though sufficient amounts of such elements are supplied ordinarily by the other constituents of the substrate medium.

A suitable basic erythromycin substrate medium ad vantageously contains from about 0.5 to about ten percent carbohydrate sources, from about 0.5 to about five percent nitrogen sources, up to about one percent nutrient salts, up to about 0.5 percent calcium carbonate or like butter salt, and the balance substantially all water. To this basic erythromycin substrate medium there is added in accordance with the invention an amount of material selected from the class consisting of propanal and n-propanol and the homologues; methanol, ethanol, and n-butanol in an amount sufficient to stimulate the production of erythromycin. Ordinarily the amount of material will not exceed about one percent. Any smaller amount down to the minimal effective concentration can be used. Ordinarily it is not desirable to use less than about 0.1 percent though. effective stimulation is sometimes obtained at lower concentrations down to about 0.05 percent. (The parts and percentages used herein are by weight unless otherwise specified.) i

The adjuvant material according to the invention advantageously is added to the erythromycin substrate medium prior to or at the time of inoculation. It can be added before the substrate medium is sterilized or sterile material can be added thereafter but prior to inoculation. The desired quantity of said adjuvant can be added all at the beginning or at periodic intervals during the fermentation. It is sometimes advantageous to add the adjuvant after the culture is well established, say from one to three days after inoculation.

Suitable adjuvants according to the invention which can be added to a basic erythromycin substrate medium to increase erythromycin production are propanal, npropanol, methanol, ethanol, and n-butanol. These materials are closely related in that the alcohols are straight chain l-alkanols containing not more than four carbon atoms and propanal in its hydrated form is the straight chain alkan-1,l-diol, propan-l,1-diol. All these materials have been observed to stimulate production of erythromycin. As will be shown, however, n-propauol is uniquely superior to the others.

The invention may now be more fully understood by the following examples which are illustrative of the processes and'composition of the present invention, but are not to be construed as limiting.

EXAMPLE 1 A sporulated culture is produced by growing Streptomyces erythreus strain NRRL 2338 on a nutrient agar slant. The spores are recovered in water suspension and used to inoculate a substrate medium suitable for producing a vegetative growth. The spore suspension is introduced into a SOO-mil flask containing mils of the following sterile substrate medium:

Dextrose (technical grade) grams 10 Beef extract do 10 Sodium chloride do 5 Bacto-peptone do 5 Water mils 1000 and incubated at 28.5 degrees Centigrade for three days t V t a 3 following erythromycin substrate medium in the portions indicated in the table:

pro-

In the following table the various adjuvants were added after sterilization but prior to seeding.

, Table II EFFECT OF VARIOUS ADJUVANTS ON YIELD OF V ERYTHROMYCIN Concentra- Peak Yield Adjuvant tion in in Micropercent grams/mil 340 0.4 455 n-Propanol 0. 4 938 n-Butanol. 0. 4 468 PropanaL- 0. 4 390 In all cases listed in the tables the initial pH was about 6.6.

It is to be understood that the invention is not limited to the exact details of operation or exact compounds shown and described as obvious modifications and equivalents will be apparent to one skilled in the art and the invention is therefore to be limited only by the scope of the appended claims.

We claim:

1. In the manufacture of erythromycin by the fermentation culturing of Streptomyces erythreus, the method which comprises culturing Streptomyces erythreus in an erythromycin substrate medium containing about 0.05 to about one percent of a substance selected from the class I a T 2,808,363

consisting of propanal, n-propanol, methanol, ethanol, and n-butanol.

2. In the manufacture of erythromycin by the fermentation culturing of Streptomyces erythreus in a substrate medium containing assimilable carbohydrate, protein, and minerals, the improvement which comprises adding to saidsubstrate medium a stimulating amount greater than about 0.05 percent of a substance selected from the class consisting of propanal', n-propanol, methanol, ethanol, and n-butanol. V V I i 3. In the manufacture of erythromycin by the fermentation culturing of Streptomyces erythreus, the method which comprises culturing Streptomyces elythreus in an erythromycin substrate medium containing about 0.05 to about one percent of n-propanol.v

4. In the manufacture of erythromycin by the fermentation culturing of Streptomyces erythreus in a substrate medium containing assimilable carbohydrate, protein, and minerals, the improvement which comprises adding to said substrate medium a stimulating amount greater than about 0.05 percent of n-propanol.

5. Inthe manufacture of erythromycin by the fermentation culturing of Streptomyces erythreus, the method which comprises culturing Streptomyces erythreus in an erythromycin substrate medium containing about 0.05 to about one percent of a straight chain 1-alkanol containing not more than four carbon atoms.

6. In the manufacture of erythromycin by the fermentation culturing of Streptomyces erythreus, the method which comprises culturing Streptomyces erythreus in an erythromycin substrate medium containing about 0.05 to about one percent of propanal.

7. In the manufacture of erythromycin by the fermena tation culturing of Streptomcyes erythreus in a substrate medium containing assimilable carbohydrate, protein, and minerals, the improvement which comprises adding to said substrate medium a stimulating amount greater than about 0.05 percent of a straight chain l alkanol containing not more than four carbon atoms.

References Cited in the file of this patent UNITED STATES PATENTS 2,653,899 Bunch Sept. 29, 1953 OTHER REFERENCES Levine et al.: Compilation of Culture Media, Williams- Wilkins, 1930, pages 14, 15, 445, 446 and 510.

Porter: Bacterial Chemistry and Physiology, Wiley, 1946, pages 799 to 800. 

1. IN THE MANUFACTURE OF ERYTHROMYCIN BY THE FERMENTATION CULTURING OF STREPTONMYCES ERYTHREUS, THE METHOD WHICH COMPRISES CULTURING STREPTOMYCES ERYTHREUS IN AN ERYTHROMYCIN SUBSTRATE MEDIUM CONTAINING ABOUT 0.05 TO ABOUT ONE PERCENT OF A SUBSTANCE SELECTED FROM THE CLASS CONSISTING OF PROPANAL, N-PROPANOL, METHANOL, ETHANOL, AND N-BUTANOL, 